DNA-protein cross-links (DPCs) are super-bulky, helix-distorting DNA lesions that result from exposure to a variety of chemical and physical agents such as antitumor drugs, environmental/ occupational toxins, ionizing radiation, and endogenous free radical-generating systems. Due to their unusually bulky nature, DPCs are hypothesized to interfere with normal DNA-protein interactions required for DNA replication, transcription, and repair, potentially leading to mutagenesis, genotoxicity, and cytotoxicity. However, the biological outcomes of DPC lesions in cells are poorly understood because of their inherent structural complexity and the difficulty of generating DNA substrates containing structurally defined DPC for biological evaluation. Our long-term goal is to elucidate the role tha spontaneous and induced DPCs play in human disease and in the anticancer activity of common chemotherapeutic agents. The objectives of this application are to determine the biological consequences of DPC formation in human cells and to discover the mechanism(s) responsible for the removal of DPCs from DNA. Our studies will for the first time systematically examine the influence of DPCs on DNA repair and replication. Our central hypothesis is that, if not repaired, DPCs exert mutagenic and cytotoxic effects, significantly contributing to the biological effects of many known carcinogens and antitumor drugs. Collectively, these studies will provide significant new insights into the molecular and cellular biology of DPCs and are expected to fundamentally advance the fields of DNA repair, cancer biology, and molecular mechanisms of aging.